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The diabetes drugs rosiglitazone and pioglitazone once looked promising for Alzheimer’s disease, then lost their luster when rosiglitazone failed in Phase 3. But have reports of their death been exaggerated? Researchers are still deciphering how they benefit cognition in AD model mice. Now, two papers shed new light on possible mechanisms. In the November 28 Journal of Neuroscience, researchers led by Michael Heneka at the University of Bonn, Germany, in collaboration with researchers at Dainippon Sumitomo Pharma, Osaka, Japan, show that pioglitazone and related drugs activate microglia to chew up Aβ deposits. Meanwhile, in the November 21 issue of the same journal, researchers led by Kelly Dineley at the University of Texas Medical Branch, Galveston, report that in AD, but not wild-type, mice, rosiglitazone kicks off a signaling pathway critical for learning and memory. This may be a compensatory mechanism to restore damaged cognition, Dineley suggested. Both Heneka and Dineley believe the pathways co-opted by rosiglitazone and pioglitazone still have potential, although it may take more brain-penetrant versions to fully exploit them. Dainippon Sumitomo Pharmaceuticals develops a similar, novel compound (DSP-8658), which is in Phase 1 clinical trials for AD, according to their 2012 second quarter report.

Olivier Thibault at the University of Kentucky, Lexington, agrees that the drugs deserve further study for AD. “These two papers present new mechanisms that may help move us toward therapeutic strategies,” he told Alzforum. He noted that, although pioglitazone and rosiglitazone bind to the same receptor, they do not turn on the same suite of genes. Therefore, drugs in this group are not all equivalent.

Rosiglitazone and pioglitazone belong to the thiazolidinediones. They bind to the nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ), which forms a heterodimer with retinoid X receptor (RXR) to affect gene transcription. Intriguingly, the RXR agonist bexarotene was recently shown to boost ApoE levels and lower Aβ (see ARF related news story; ARF related news story). Activation of PPARγ signaling by thiazolidinediones is known to sensitize tissues to insulin and to dampen inflammation, and this caught the interest of AD researchers (see ARF Webinar). Pioglitazone and rosiglitazone have also been shown to lower Aβ deposits (see Searcy et al., 2012; Escribano et al., 2010). However, the drugs, particularly rosiglitazone, cause many side effects, such as cardiovascular problems and weight gain (see, e.g., ARF related news story). Both drugs hardly cross the blood-brain barrier, as they were not initially designed to act on the central nervous system. Rosiglitazone showed hints of cognitive benefit in Phase 2 trials but failed in Phase 3 (see ARF related news story).

To test this, first author Mitsugu Yamanaka added synthetic Aβ42 to rat primary microglial cultures, along with either pioglitazone or the novel PPARα/γ agonist, DSP-8658. The latter penetrates the brain better than do thiazolidinediones. Over four hours, treated microglia took up about twice as much Aβ as did control cells. Silencing PPARγ abolished the effect, showing that it occurs through this pathway. The authors also stimulated phagocytosis with the RXR agonists bexarotene and retinoic acid. Adding PPARγ and RXR agonists together produced an additive effect, allowing microglia to engulf up to four times as much Aβ as did unstimulated cells.

Looking for the mechanism behind the improved phagocytosis, the authors uncovered an essential role for the receptor CD36, which microglia use to bind material they engulf. PPARγ activation turns on CD36 (see Tontonoz et al., 1998). Blocking the receptor, or protein transcription or translation in general, abolished the beneficial effect of PPARγ agonists. This suggests that the pathway dials up CD36 expression, enabling microglia to devour Aβ.

To see if the results held in vivo, the authors fed DSP-8658 to four-month-old APPPS1 mice. After two months, treated mice had 30 percent more Aβ-containing microglia, 30 percent less soluble Aβ40 and Aβ42, and half the plaque area than did untreated transgenics. Treated mice also performed better than controls in the Morris water maze.

The findings emphasize the role inflammation and sluggish phagocytosis play in late-onset AD, Heneka said. Sporadic AD is believed to result from inefficient clearance of Aβ, rather than enhanced production (see, e.g., ARF related news story). Intriguingly, mutations in TREM2, another microglial receptor involved in phagocytosis, were recently shown to be potent genetic risk factors (see ARF related news story). “I think we will see more inflammatory or innate immune genes [as risk factors],” Heneka predicted.

Heneka noted that newer drugs such as DSP-8658 are safer and more brain penetrant than rosiglitazone and pioglitazone, and show potential for future clinical applications. However, he thinks PPARγ agonists will be most effective in MCI or even earlier, as chronic inflammation shuts down the PPARγ pathway in later stages of AD. More preclinical work will be needed before taking these agents to AD trials, he added. DSP-8658 completed a Phase 1 trial for diabetes.

Marie-Eve Tremblay at the University of Wisconsin, Madison, noted that work from her group and others' has shown that microglia prune synapses (see ARF related news story). Synapse loss correlates well with cognitive impairment in AD. “Does DSP-8658 also influence the phagocytosis of synapses in this model? Importantly, answering this question could provide novel insights into the mechanisms underlying the loss of synapses in AD,” she wrote to Alzforum (see full comment below).

First author Larry Denner turned to proteomics to find out. He fed the drug to eight-month-old Tg2576 mice for one month, then used quantitative mass spectrometry to compare protein levels in the dentate gyrus of treated and untreated mice. Rosiglitazone induced many proteins involved in synaptic plasticity and memory that are also regulated by ERK/MAPK. The drug normalized the expression, activation, and nuclear localization of hippocampal PPARγ, and boosted nuclear ERK activity compared to untreated animals. Injecting a PPARγ antagonist into the brain ventricles blocked these effects and also abolished the cognitive improvement normally seen with rosiglitazone, showing that the effects go through this pathway. Importantly, neither rosiglitazone nor the PPARγ antagonist affected PPARγ, ERK, or cognition in wild-type mice, supporting the idea that this pathway only becomes activated in diseased brains as a compensatory mechanism. Rosiglitazone treatment did not affect Aβ accumulation, the authors report.

Since then, the authors have gleaned some clues to how PPARγ might recruit ERK/MAPK pathways. As described by first author Jordan Jahrling in a poster at the Society for Neuroscience 42nd annual conference, held 13-17 October in New Orleans, Louisiana, PPARγ forms a complex with activated (phosphorylated) ERK. In Tg2576 mice, higher levels of this complex in hippocampal nuclei correlated with better cognition, and rosiglitazone treatment further boosted levels. PPARγ-pERK complexes occurred in wild-type mice as well, but did not increase with drug, Jahrling reported. Human postmortem hippocampi also contain the complexes. In a half-dozen AD brains, higher PPARγ-pERK levels associated with better scores those people had had on the Mini-Mental State Exam, while in three healthy controls the complex did not relate to cognition.

Together, the evidence suggests that rosiglitazone harnesses the dysregulated ERK/MAPK cascade in AD brains to restore cognition, Dineley said. “It resets the dynamic range that is necessary for synaptic plasticity, learning, and memory,” she explained. Dineley is collaborating with Patrick Griffin at the Scripps Research Institute, Jupiter, Florida, to test alternative PPARγ ligands with fewer side effects to see if they provide cognitive benefits. Like Heneka, Dineley believes PPARγ agonists would be effective at the MCI stage or earlier.—Madolyn Bowman Rogers

In Alzheimer’s disease, synapse loss best correlates with the progressive impairment in learning and memory, even though amyloid-β plaques and neurofibrillary tangles of hyperphosphorylated tau are the most prominent hallmarks. Yamanaka et al. reveal that microglial phagocytosis of amyloid-β induced by PPARγ/RXRα activation improves spatial learning and memory in the APPPS1 mouse model. The PPARγ activator DSP-8658 had similar effects.

Is microglial phagocytosis of amyloid-β specifically targeted by the DSP-8658, or does the drug also influence the phagocytosis of synapses in this model? Importantly, answering this question could provide novel insights into the mechanisms underlying the loss of synapses in relation to the hallmarks of Alzheimer’s disease.

This interesting work from Michael Heneka’s group adds to a growing body of evidence bolstering the potential of PPARγ agonists for the treatment of AD. Even more encouraging, the authors’ PPARγ agonist of choice, DSP-8658, is already in developmental trials for treatment of type 2 diabetes and has shown a favorable safety profile so far. The current authors have gone further, though, by also making a foray into mechanistic biology. Specifically, they have nicely shown that PPARγ stimulation promotes microglial Aβ phagocytosis via the innate immune scavenger receptor, CD36. These beneficial effects on Aβ uptake were further augmented by combined agonism of PPARγ and retinoid X receptors. Finally, treatment of the PSAPP mouse model of accelerated cerebral amyloidosis led to an increase in Aβ phagocytosis by microglia in vivo, mitigation of cerebral amyloidosis, and improvement of cognitive impairment. If this mouse model is representative of the clinical syndrome, then the translational potential of PPARγ agonism is certainly something to be excited about.

Even though they are members of the same drug class and share properties, it is also no surprise that pioglitazone and rosiglitazone have disparate effects, as demonstrated by the Aβ results highlighted in the recent publications from the Heneka and the Dineley groups. In fact, we previously showed that pioglitazone and rosiglitazone target different calcium influx pathways (GluRs and VGCCs, respectively) in hippocampal neurons (Pancani et al., 2009). Further, it is well appreciated that they have different effects and safety profiles in the cardiovascular system. Future studies directly comparing the genomic and/or proteomic targets will help parse out the underlying mechanisms responsible for the differences seen with these two drugs.

<p>The proteomic analysis of rosiglitazone actions in the dentate gyrus presented by Dr. Dineley’s group highlighted the ERK/MAPK pathways as a central target. Their results corroborate our prior microarray analysis of hippocampal genes sensitive to pioglitazone in 3xTg AD mice (Searcy et al., 2012). Gene pathways decreased by chronic pioglitazone treatment included synaptic structure and energy metabolism, as well as some inflammatory processes. Conversely, those increased included cellular assembly and biosynthetic processes. Of note, we also identified female hormone/estrogen and glutamatergic neurotransmission as processes targeted by pioglitazone. These processes are also associated with ERK/MAPK signaling and, importantly, with mechanisms of memory formation and recall. Lending support to Dr. Tremblay’s comment above, our work also showed that synaptic communication (throughput and LTP) was also enhanced by pioglitazone, restoring a phenotype typically seen in younger animals.

</p><p>Finally, it is encouraging to see the development and promising results with a more brain-permeant PPAR-γ agonist (DSP-865, see Heneka paper). Time will tell if this compound will help unify PPAR-γ agonist mechanisms in the brain. Irrespective, an increase in brain permeability is likely to have an important impact on CNS outcome and may also help reduce peripheral side effects.